Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling.

Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size.

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

Structural Basis of Smoothened Activation in Hedgehog Signaling.

The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.

Dynamic BTB-domain filaments promote clustering of ZBTB proteins.

The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.

Mechanisms of Mitotic Spindle Assembly.

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.

Amyloid-like Self-Assembly of a Cellular Compartment.

Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells.

Mechanism of NMDA Receptor Inhibition and Activation.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that mediate synaptic transmission and underpin learning and memory. NMDAR dysfunction is directly implicated in diseases ranging from seizure to ischemia. Despite its fundamental importance, little is known about how the NMDAR transitions between inactive and active states and how small molecules inhibit or activate ion channel gating. Here, we report electron cryo-microscopy structures of the GluN1-GluN2B NMDA receptor in an ensemble of competitive antagonist-bound states, an agonist-bound form, and a state bound with agonists and the allosteric inhibitor Ro25-6981. Together with double electron-electron resonance experiments, we show how competitive antagonists rupture the ligand binding domain (LBD) gating "ring," how agonists retain the ring in a dimer-of-dimers configuration, and how allosteric inhibitors, acting within the amino terminal domain, further stabilize the LBD layer. These studies illuminate how the LBD gating ring is fundamental to signal transduction and gating in NMDARs.

Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility.

Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications.

GM130 Regulates Golgi-Derived Spindle Assembly by Activating TPX2 and Capturing Microtubules.

Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and organize microtubules in a precise spatiotemporal manner. Among them, the contributions of centrosomes, chromosomes, and microtubules have been well studied, yet the involvement of membrane-bound organelles remains largely elusive. Here, we provide mechanistic evidence for a membrane-based, Golgi-derived microtubule assembly pathway in mitosis. Upon mitotic entry, the Golgi matrix protein GM130 interacts with importin α via a classical nuclear localization signal that recruits importin α to the Golgi membranes. Sequestration of importin α by GM130 liberates the spindle assembly factor TPX2, which activates Aurora-A kinase and stimulates local microtubule nucleation. Upon filament assembly, nascent microtubules are further captured by GM130, thus linking Golgi membranes to the spindle. Our results reveal an active role for the Golgi in regulating spindle formation to ensure faithful organelle inheritance.

A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

Single-strand DNA breaks cause replisome disassembly.

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks," which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress.

Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin.

The BRCA1-BARD1 complex directs the DNA double-strand break (DSB) repair pathway choice to error-free homologous recombination (HR) during the S-G2 stages. Targeting BRCA1-BARD1 to DSB-proximal sites requires BARD1-mediated nucleosome interaction and histone mark recognition. Here, we report the cryo-EM structure of BARD1 bound to a ubiquitinated nucleosome core particle (NCPUb) at 3.1 Å resolution and illustrate how BARD1 simultaneously recognizes the DNA damage-induced mark H2AK15ub and DNA replication-associated mark H4K20me0 on the nucleosome. In vitro and in vivo analyses reveal that the BARD1-NCPUb complex is stabilized by BARD1-nucleosome interaction, BARD1-ubiquitin interaction, and BARD1 ARD domain-BARD1 BRCT domain interaction, and abrogating these interactions is detrimental to HR activity. We further identify multiple disease-causing BARD1 mutations that disrupt BARD1-NCPUb interactions and hence impair HR. Together, this study elucidates the mechanism of BRCA1-BARD1 complex recruitment and retention by DSB-flanking nucleosomes and sheds important light on cancer therapeutic avenues.

γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/ß-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.

Scaling of dorsal-ventral patterning by embryo size-dependent degradation of Spemann's organizer signals.

Spemann's organizer plays a key role in dorsal-ventral (DV) patterning in the amphibian embryo by secreting diffusible proteins such as Chordin, an antagonist to ventralizing bone morphogenetic proteins (BMPs). The DV patterning is so robust that an amphibian embryo with its ventral half surgically removed can develop into a smaller but proportionally patterned larva. Here, we show that this robust patterning depends on facilitated Chordin degradation and requires the expression of the Chordin-proteinase inhibitor Sizzled on the opposite side. Sizzled, which is stable and diffuses widely along the DV axis, stabilizes Chordin and expands its distribution in the ventral direction. This expanded Chordin distribution, in turn, limits BMP-dependent Sizzled production, forming an axis-wide feedback loop for shaping Chordin's activity. Using bisection assays, we demonstrate that Chordin degradation is dynamically controlled by embryo-size-coupled Sizzled accumulation. We propose a scaling model that enables the DV pattern to adjust proportionally to embryonic axis size.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2.

The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.

Zygotic genome activation during the maternal-to-zygotic transition.

Embryogenesis depends on a highly coordinated cascade of genetically encoded events. In animals, maternal factors contributed by the egg cytoplasm initially control development, whereas the zygotic nuclear genome is quiescent. Subsequently, the genome is activated, embryonic gene products are mobilized, and maternal factors are cleared. This transfer of developmental control is called the maternal-to-zygotic transition (MZT). In this review, we discuss recent advances toward understanding the scope, timing, and mechanisms that underlie zygotic genome activation at the MZT in animals. We describe high-throughput techniques to measure the embryonic transcriptome and explore how regulation of the cell cycle, chromatin, and transcription factors together elicits specific patterns of embryonic gene expression. Finally, we illustrate the interplay between zygotic transcription and maternal clearance and show how these two activities combine to reprogram two terminally differentiated gametes into a totipotent embryo.

A Mechanism to Minimize Errors during Non-homologous End Joining.

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires the formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.

The DNA replication fork suppresses CMG unloading from chromatin before termination.

When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.

SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

BET activity plays an essential role in control of stem cell attributes in Xenopus.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.